Peptide derivative fusion inhibitors of hiv infection

ABSTRACT

This invention relates to gp41 peptide derivatives that are inhibitors of viral infection and/or exhibit antifusogenic properties. In particular, this invention relates to gp41 derivatives having inhibiting activity against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) with enhanced duration of action for the treatment of the respective viral infections.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to human immunodeficiency virus (hereinafter“HIV”) gp41 C-terminal peptide derivatives that are inhibitors of viralinfection and/or exhibit antifusogenic properties. In particular, thisinvention relates to peptide derivatives having inhibiting activityagainst HIV and simian immunodeficiency virus (hereinafter “SIV”), withimproved solubility and extended duration of action for the treatment ofthe respective viral infections.

2. Review of Related Art

Membrane fusion events are commonplace in normal cell biologicalprocesses, and membrane fusion is also involved in a variety of diseasestates, including, for example the entry of enveloped viruses intocells. Some enveloped viruses fuse with target cells by specific bindingreactions between proteins of the virus envelop and cell surfaceproteins which trigger conformational changes in associated viralproteins that in turn promote fusion of the viral envelop with the cellmembrane.

One enveloped virus, HIV, is a member of the lentivirus family ofretroviruses, and there are two prevalent types of HIV, HIV-1 and HIV-2,with various strains of each having been identified. The fusion of HIVand its host cells is mediated by the binding of viral envelop proteinsgp120 and gp41, with the CD4 glycoprotein and a chemokine co-receptor onthe cell surface. Binding of gp120 to CD4 on the surface of T cells andto a co-receptor (e.g., CCR5 or CXCR4) is followed by insertion of gp41into the membrane of the target cell; then helicies from the N-terminalportion of gp41 form coiled coil structures with helicies from theC-terminal portion of the same protein, which draws the virus and thecell together for fusion (Malashkevich, et al., Proc. Natl. Acad. Sci.USA, 1998 August 4; 95(16):9134-9).

Peptides are known to inhibit or otherwise disrupt membranefusion-associated events, including, for example, inhibiting retroviraltransmission to uninfected cells. Peptides from the second heptad repeatregion of HIV envelop protein gp41, including T20 (DP178) and C34, haveshown potent anti-viral activity against HIV in vitro (see Wild, et al.,1994, Proc. Natl. Acad. Sci. USA, 91:9770-4; Chan, et al., 1998, Proc.Natl. Acad. Sci. USA, 95:15613-15617). The demonstrated anti-viralactivity includes inhibiting CD4⁺ cell infection by free virus and/orinhibiting HIV-induced syncytia formation between infected anduninfected CD4⁺ cells. The inhibition is believed to occur by binding ofthese peptides to the first heptad repeat region in gp41, thuspreventing the first and second heptad repeat regions from forming thefusogenic hairpin structure.

While many of the anti-viral or anti-fusogenic peptides described in theart exhibit potent anti-viral and/or anti-fusogenic activity in vitro,they suffer from short half-life in vivo, primarily due to rapid serumclearance and peptidase and protease activity. This in turn greatlyreduces their effective anti-viral activity. There is therefore a needfor a method of prolonging the half-life of peptides in vivo withoutsubstantially affecting the anti-fusogenic activity.

One method for prolonging the half-life of peptides is disclosed in U.S.Pat. No. 5,612,034, which describes a method for covalently coupling atherapeutic peptide to a native protein found in the blood stream. Thepeptide is modified with a chemically reactive moiety that is capable ofreacting with fuctionalities present on proteins in the blood stream.Upon injection of the modified peptide into the blood stream, it islinked to a long-lived blood component forming a long-lived depot of thepeptide. However, since the molecular weight of proteins in the bloodstream ranges between 50-600 kD, there is concern that the biologicalactivity of such linked peptides may be compromised by steric hinderanceof the much larger size protein.

An attempt to prolong the half-life of a known anti-fusogenic peptide isdisclosed in International Patent Publication WO 00/69902 (hereinafter“the '902 publication”) by Conjuchem, Inc. In this disclosure, DP178 ismodified by attaching 3-maleimidopropionic acid by an amide link to theepsilon amino group of lysine which is in turn linked by peptide bond tothe C-terminal Phe of DP178. The '902 publication also proposes analogsof the modified DP178 which are either truncations of DP178 orcorresponding fragments of gp41 from other HIV viral isolates. The '902publication does not suggest any other design criteria foranti-fusogenic peptides.

Therefore, there remains a need for a method of prolonging the half-lifeof peptides in vivo without substantially affecting the anti-fusogenicactivity.

SUMMARY OF THE INVENTION

The present invention is directed to HIV gp41 peptide derivatives havinganti-viral, virostatic and/or anti-fusogenic activity, including but notlimited to the modified peptides of Tables 1, 2 and 3 and FIG. 1, aswell as modified and derivatized forms thereof (hereinafter collectivelyreferred to as “variant gp41 peptides”). These variant gp41 peptidesprovide for an increased in vivo stability and a reduced susceptibilityto peptidase or protease degradation. As a result, the variant gp41peptides minimize the need for more frequent, or even continual,administration as would be expected with unmodified HIV gp41 peptides.The present peptide derivatives, and derivatives made using methods ofthe invention for gp41-like sequences from other viruses, can be used,e.g., as a prophylactic against and/or treatment for infection of anumber of viruses, including but not limited to HIV and SW.

In accordance with the present invention, there are now provided peptidederivatives having enhanced solubility and antiviral activity whencompared with the corresponding unmodified peptide sequence of HIV gp41.More specifically, the present invention is concerned with compounds ofthe formulas illustrated in Tables 1, 2 and 3 and FIG. 1 infra, whichinclude peptide derivatives capable of reacting with thiol groups on ablood component, either in vivo or ex viva, to form a stable covalentbond.

TABLE 1  Peptide Fragments of gp41 and Modified AnalogsAc-SLEQIWNNMT WEEWDREINN YTELIHELIE ESQNQQEKNE QELL-NH2 (SEQ ID NO: 1)                        FB005Ac-WEEWDREINN YTKLIHELIE ESQNQQEKNE QELL-NH2 (SEQ ID NO: 2)                        FB006Ac-WEEWDREINN YTKLIHELIE ESQNQQEENE QELL-NH2 (SEQ ID NO: 7)                        FB066AC-WQE WEQKITALLE QAQICREKNE YELQKLDKWA SLWEWF-NH2 (SEQ ID NO: 3)                        T-1249Ac-YTSLIHSLIE ESQNQQEKNE QELLELDKWA SLWNWF-NH2 (SEQ ID NO: 4)                         T-20Ac-WMEWDREINN YTSLIHSLIE ESQNQQEKNE QELL-NH2 (SEQ ID NO: 5)                         C-34

TABLE 2 Maleimide Modified Peptides

  FB005M (SEQ ID NO: 8)

  FB005CM (SEQ ID NO: 9)

  FB006M (SEQ ID NO: 10)

  FB007M (SEQ ID NO: 11)

  FB010M (SEQ ID NO: 12)

  FB010KM (SEQ ID NO: 13)

  FB066M (SEQ ID NO: 14)

  FB066KM (SEQ ID NO: 15)

This invention provides novel compositions, containing peptides havingmodification of predetermined residues (i.e., point mutations) relativeto the native peptide which are introduced to improve activity andsolubility. The predetermined residues consist of the underlined aminoacid residues of the peptide sequences found in Table 3. The peptideshaving modified residues include, but are not limited to, substitutedamino acid residues wherein amino acid residues having either theproperties of increased hydrophilic or hydrophobicity are substitutedfor native amino acid residues. The variant gp41 peptides may also besubstituted with amino acid residues having high alpha helical-formingpropensities. Alternatively, the peptides having modified residuesinclude, but are not limited to, derivatized amino acid residues whereina coupling group is conjugated to a pre-determined amino acid residue,thereby allowing covalent bonding of the derivatized peptide to a bloodcomponent.

In another aspect, this invention provides pharmaceutical compositionscomprising the derivatives of the above formulae in combination with apharmaceutically acceptable carrier. Such compositions are useful forinhibiting the activity of HIV (including HIV-1, HIV-2 and all serotypesthereof) and SW.

In a further embodiment of the present invention, there is provided amethod for inhibiting the infection of HIV or SW. The method comprisesadministering to a subject, preferably a mammal, and most preferably ahuman, a virus-inhibiting effective amount of one or more variant gp41peptides, alone or in combination with a pharmaceutical carrier, or incombination with other antiviral agents including other variant gp41peptides. In a particularly preferred embodiment of the invention, atleast one of the variant gp41 peptides, alone or in combination with apharmaceutical carrier, or in combination with other antiviral agentsincluding other variant gp41 peptides, may be administered to a subjectin a virus-inhibiting amount.

In a further aspect of the present invention, there is provided aconjugate comprising at least one of the variant gp41 peptidescovalently bonded to a blood component. In one embodiment of theinvention, preferred blood components for reaction with the compounds ofthis invention include proteins such as immunoglobulins, including IgGand IgM, serum albumin, ferritin, steroid binding proteins, transferrin,thyroxin binding protein, α-2-macroglobulin etc., serum albumin and IgGbeing a more preferred embodiment, and serum albumin being the mostpreferred embodiment of the invention.

In a further aspect of the present invention, there is provided a methodfor extending the in vivo half-life of the variant gp41 peptides in asubject, the method comprising covalently bonding one or more of thevariant gp41 peptides to a blood component.

In another embodiment of the invention, a method is provided for thedesign, synthesis and testing of novel peptides having anti-viral,virostatic or anti-fusogenic activity against a variety of viruses. Themethod involves screening of viral proteins involved with cellular entryto identify peptide sequences therein harboring alpha-helical formingpropensities, and designing compositions based off of these peptidesthat can be used to treat the diseases caused by the same viruses. Themethod also contemplates in vitro testing of the peptide compositions toverify anti-viral, virostatic or anti-fusogenic activity.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1—FIG. 1 shows the aligned sequences of various peptides disclosedin the present invention.

DETAILED DESCRIPTION OF TILE INVENTION

As used herein, “derivatization” shall mean the addition of couplinggroups to peptide sequences. Representative coupling groups are moreparticularly provided infra.

As used herein, “modification” shall mean the substitution of a firstamino acid in a native peptide sequence by a second amino acid. Thesecond amino acid may be selected from the non-limiting group ofhydrophilic amino acids, hydrophobic amino acids, amino acids havinghelical propensities, non-naturally occurring amino acids and theD-isomers of the naturally occurring L-amino acids.

Fusion of HIV-1 and related lentiviruses with target cells can beinhibited by peptide fragments of the native viral envelop proteinswhich accomplish the fusion. These peptide fragments can bind to theenvelop proteins and block binding of distal portions of the viralenvelope proteins, thereby inhibiting conformational changes in thenative protein that are critical to effect the fusion of HIV-1 to targetcells. These peptides, by blocking fusion of the virus with the cells,interrupt the infectious process necessary for disease progression.

The present invention improves on the properties of existing anti-viraland anti-fusogenic peptides and provides novel peptide compositionsuseful to treat HIV and SIV. The viruses that may be inhibited by thepeptides of this invention include, but are not limited to, the humanretrovirus HIV (including HIV-1 and HIV-2, as well as all otherserotypes thereof) and SIV.

Modified Peptides

Modified, derivatized peptides with anti-fusogenic activity againstlentiviruses can be prepared according to this invention. Theanti-fusogenic peptides are helix-forming peptides based on native gp41protein sequence, which are modified by changing selected amino acids ofthe peptides. The modified amino acids are selected to avoid disruptingthe interactions which contribute to the formation of coiled-coilcomplexes with helicies of viral envelop protein gp41. In oneembodiment, the amino acid residues selected for modification are thosewhose side chains are away from the coiled-coil interface. Theseresidues are substituted with alternative residues that will enhanceeither the hydrophobic or hydrophilic properties of the peptides, oralternatively are derivatized to provide reactive moieties that enablecovalent bonding of the peptides to circulating blood proteins. Theintroduction of hydrophilic residues into a peptide sequence willincrease the solubility of the peptide. The introduction of hydrophobicresidues into a peptide sequence will decrease the solubility of thepeptide. In one embodiment of the invention, modified peptides includethe peptides designated FB005, FB006 and FB066, and especiallyderivatives of these peptides with maleimide coupling moieties, such as3-maleimidopropionic acid coupled to lysine through [2-(2-amino-ethoxy)ethoxy]acetic acid, or other equivalent coupling structures. In anotherembodiment of the invention, amino acids in the peptide sequence aresubstituted with amino acids having a propensity to form alpha-helices.

Alternatively, chemical groups can be added at their amino and/orcarboxy termini, such that for example, the stability, reactivity and/orsolubility of the peptides is enhanced. For example, hydrophobic groupssuch as carbobenzoxyl, dansyl, acetyl or t-butyloxycarbonyl groups, maybe added to the peptides' amino termini. Likewise, an acetyl group or a9-fluorenylmethoxy-carbonyl group may be placed at the peptides' aminotermini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or anamido group may be added to the peptides' carboxy termini. Similarly, apara-nitrobenzyl ester group may be placed at the peptides' carboxytermini. Techniques for introducing such modifications are well known tothose of skill in the art.

The peptides may be synthesized such that their steric configuration isaltered. For example, the D-isomer of one or more of the amino acidresidues of the peptide may be used, rather than the usual L-isomer. Inone embodiment of the invention, at least two or more amino acidsubstitutions comprise D-isomers of the naturally occurring L-aminoacids. In another embodiment of the invention, each of the naturallyoccurring L-amino acids in the complete peptide sequence is substitutedwith a D-isomer of the same amino acid. The invention also contemplatesthat at least one of the amino acid residues of the variant gp41peptides may be substituted by one of the well known non-naturallyoccurring amino acid residues. In another embodiment of the invention,any combination of substitutions of the D-isomers of the naturallyoccurring L-amino acids, or non-naturally occurring amino acids, may bemade to the variant gp41 peptides. Alterations such as these may serveto increase the stability, protease-resistance, activity, reactivityand/or solubility of the variant gp41 peptides.

Non-naturally occurring amino acids are well known in the art.Furthermore, methods of synthesizing peptides having either D-isomers ofthe naturally occurring L-amino acids or non-naturally occurring aminoacids are also well known in the art (See, for example, the disclosuresof U.S. Pat. Nos. 5,840,697 and 6,268,479, as well as Biochemistry(Chap. 4), D. Voet and J. G. Voet, Wiley & Sons (1990), which are hereinincorporated by reference), and are also within the contemplation ofthis invention.

In one embodiment of the invention, modified peptides include thepeptides designated FB005, FB006 and FB066, and especially derivativesof these peptides with maleimide coupling moieties, such as3-maleimidopropionic acid coupled to lysine through[2-(2-amino-ethoxy)ethoxy]acetic acid, or other equivalent couplingstructures.

The invention further encompasses variant gp41 peptides wherein aminoacid residues thereof are substituted with either hydrophilic orhydrophobic residues, thereby altering the aqueous traits of thepeptides. Alternatively, other amino acid residues of the variant gp41peptides are derivatized with a maleimide linking moiety. In a preferredembodiment of the invention, the underlined amino acid residues in thefollowing variant gp41 peptides (presented in Table 3) are substitutedwith hydrophilic or hydrophobic residues, or alternatively arederivatized with a maleimide linking moiety. Any other peptidesencompassed by this invention having a C-terminal lysine residue mayalso have that C-terminal lysine residue substituted with hydrophilicresidues, or alternatively derivatized with a maleimide linking moiety:

TABLE 3  YT S LIHSLI E ESQNQQ E KNEQEL L ELDKWA S LWNWF (SEQ ID NO: 4)WQEWE Q KITALL E QAQIQQ E KNEYEL Q KLDKWA S LWEWF (SEQ ID NO: 3) WMEWD REINNYT S LIHSLIL E SQNQQ EK NEQEL L (SEQ ID NO: 5) WQEWE R KVDFLE ENITALL E EAQIQQ EK NMYEL Q (SEQ ID NO: 6) SLEQIWNNMTWEEWD R EINNYT ELIHELI E ESQNQQ E KNEQEL L (SEQ ID NO: 1) WEEWD R EINNYT K LIHELIEESQNQQ EK NEQEL L (SEQ ID NO: 2) WEEWD R EINNYT K LIHELI E ESQNQQ EENEQEL L (SEQ ID NO: 7) SLEQIWNNMTWEEWD R EINNYT X LIHELI E ESQNQQ EKNEQEL L (SEQ ID NO: 8) SLEQIWNNMTWEEWD R EINNYT E LIHELI E ESQNQQ EKNEQEL L X (SEQ ID NO: 9) WEEWD R EINNYT X LIHELI E ESQNQQ EK NEWEL L(SEQ ID NO: 10) WEEWD R EINNYT E LIHELI E ESQNQQ EK NEQEL L X(SEQ ID NO: 11) WQEWE Q KITALL X QAQIQQ E KNEYEL Q KLDKWA S LWEWF(SEQ ID NO: 12) WQEWE Q KITALI E QAQIQQ E KNEYEL Q KLDKWA S LWEWFX(SEQ ID NO: 13)

Hydrophilic amino acids which may be substituted for any of theunderlined amino acids include those amino acids listed in Table 4.

Hydrophobic amino acids which may be substituted for any of theunderlined amino acids include those amino acids listed in Table 5.

Additionally, any of the underlined amino acid residues presented inTable 3 may be derivatized with a maleimide linking moiety, therebyproviding the amino acid residue with which the variant gp41 peptide(s)may be covalently bonded to the available thiol group(s) present onblood components. In a preferred embodiment of the invention, lysineresidues are derivatized with a maleimide linking moiety. In aparticularly preferred embodiment of the invention, lysine residue(s)derivatized with a maleimide linking moiety is covalently bonded to athiol group(s) present on a blood component.

In another embodiment of the invention, any of the underlined amino acidresidues presented in Table 3 may be substituted with amino acids havinghigh helical propensity (See Creamer, T., et al., Alpha-helix-formingpropensities in peptides and proteins. Proteins, June; 19(2):85-97(1994)). Amino acids having high helical propensity are listed in Table6 in descending order of α-helical propensity. Because the activeconformation of these peptides is believed to be alpha helical whenbound to the viral target gp41, increased tendencies to form helices canpotentially increase the antiviral activity.

TABLE 4 Hydrophilic Amino Acids Amino Acid Abbreviation Arginine ArgLysine Lys Aspartic Acid Asp Glutamic Acid Glu Asparagine Asn GlutamineGln Histidine His Serine Ser Threonine Thr Glycine Gly

TABLE 5 Hydrophobic Amino Acids Amino Acid Abbreviation Alanine AlaIsoleucine Ile Leucine Leu Methionine Met Phenylalanine Phe TryptophanTrp Valine Val Tyrosine Tyr

TABLE 6 Amino Acids having high helical propensity¹ Amino Acid α-HelicalPreference Glutamic acid 1.59 Alanine 1.41 Leucine 1.34 Methionine 1.30Glutamine 1.27 Lysine 1.23 Arginine 1.21 Phenylalanine 1.16 Isoleucine1.09 Histidine 1.05 Tryptophan 1.02 Aspartic acid 0.99 Valine 0.90Threonine 0.76 Asparagine 0.76 Tyrosine 0.74 Cysteine 0.66 Serine 0.57Glycine 0.43 Proline 0.34 ¹Source: T. E. Creighton, Proteins: Structureand Molecular Properties (2^(nd) Ed.), W.H. Freeman and Co., 1993.

Generally speaking, peptides of the invention are C-34 analogscomprising five heptads of one alpha helix of a coiled coil proteincomplex, preferred analogs having maleimide coupling groups and residuesmore polar than the parent sequence substituted at residue 2 of 7 of thefirst heptad, residue 6 of 7 of the second heptad, residue 3 of 7 of thethird heptad and/or residue 7 of 7 of the fourth heptad. In anotherembodiment of the invention, peptides of the invention encompass theseabove-recited peptides, but further include an additional 10 residuesfrom gp41 introduced at the N-terminus of the C-34 peptide.

Peptide FB006 is based on the C34 peptide with the second and theseventeenth residues mutated to glutamate, and the thirteenth residuemutated to lysine. The mutation positions were selected based on thecrystal structure of the N36/C34 complex. The selection criterion isthat these residues are not involved in binding to the N36 helices.Mutations to glutamate and lysine are aimed to improve the solubilityand helical propensity, which is the tendency to form a helix in aqueoussolution. Because it is believed that the active conformation of C34 ishelical as in the N36/C34 crystal structure; enhanced helical propensitythus should improve the biological activity. Peptides FB005, FB006,FB066, FB005M, FB005CM, FB006M, and FB007M also contain thesesubstitutions.

Variant gp41 peptides encompass the peptide sequences listed in Tables1, 2 and 3, and FIG. 1, as well as modified and derivatized formsthereof. Peptide FB005 is based on the FB006 peptide, but has anadditional 10 amino acid residues located at the N-terminus relative toother variant gp41 peptides.

Peptide FB066 is based on FB006. It is different from FB006 in that itharbors a single amino acid substitution, changing the lysine atposition 28 to a glutamic acid. This change leaves the 13^(th) aminoacid residue as the only lysine residue to function as the conjugationsite. This change significantly simplifies the synthesis of analogs withmaleimide modifications.

The invention also provides derivatives based on FB005, FB006, andT-1249 (see WO 01/03723) which can conjugate with serum albumin tobecome long lasting inhibitors. Peptides FB005M and FB005CM are based onthe FB005 sequence; peptides FB006M and FB007M are based on FB006sequence; and peptides FB010M and FB010KM are based on the T-1249sequence.

The method of selecting the linkage site on the peptide to enablelinkage to the blood protein carrier is also novel. The inventors foundthat linking the variant gp41 peptide to albumin via an internal Lysineresidue of the peptide yields a conjugate with improved efficacy over aC-terminal linkage. The IC₅₀ ² for FB006, FB006M, and FB007M are 1.4,3.9 and 9.1 nM respectively. FB006 is the native peptide, FB006M is amodified peptide complex harboring a maleimide linkage at the 13^(th)residue, while FB007M is linked at the C-terminus. When FB006M is linkedto serum albumin, the amount needed for antiviral effect increases by2.8-fold while linking to albumin via the C-terminal linkage of FB007Mcauses the IC₅₀ to increase in value by 6.5-fold. Although linking to acarrier molecule was anticipated to extend the ½-life of the peptide,conceptually conjugation to albumin (a 66 kDa protein) was also expectedto block the biological activity of the peptides by providing a sterichinderance. Unexpectedly, however, when the inventors prepared FB006Mpeptides and conjugated it to albumin, it was found that the antiviralactivity of the complex was not appreciably compromised (increase only2.8-fold). ² The IC₅₀ value is the drug concentration for achieving 50%viral inhibition, and TC₅₀ value is the drug concentration for achieving50% cytotoxicity.

Coupling groups of the invention are chemical groups capable of forminga covalent bond with a functionality present on a blood component.Coupling groups are generally stable in an aqueous environment. Thereactive functionalities which are available on blood components forcovalent bonding to the coupling groups are primarily amino groups,carboxyl groups and thiol groups. In one embodiment of the invention,coupling groups include, but are not limited to, reactive double bonds,carboxy, phosphoryl, or convenient acyl groups, either as an ester or amixed anhydride, or an imidate, thereby capable of forming a covalentbond with functionalities such as amino groups, hydroxy groups or thiolgroups at the target site on mobile proteins, in particular on bloodproteins. Reactive ester coupling groups consist of phenolic compounds,thiol esters, alkyl esters, phosphate esters, or the like. In aparticularly preferred embodiment of the invention, coupling groupsconsist of succinimidyl or maleimido groups.

The focus of the present invention is to modify gp41 peptide sequencesto confer improved bio-availability, extended half-life and betterdistribution (through selective conjugation of the peptide onto aprotein carrier) to the peptides without substantially modifying theanti-viral, virostatic or anti-fusogenic properties of the peptides.Derivatization of variant gp41 peptides as described herein allows thederivatized peptides to react with groups on blood components(particularly available thiol groups) to form stable covalent bonds.Preferred derivatives of variant gp41 peptides are designed tospecifically react with thiol groups on mobile blood proteins. Suchreaction is established by covalent bonding of the peptide having amaleimide link to a thiol group on a mobile blood protein such as serumalbumin or IgG. Thus, one embodiment of the invention comprises amodified peptide covalently linked to a blood protein, including amobile blood protein. A particularly preferred embodiment of theinvention involves covalent bonding of the modified peptide to serumalbumin.

The blood components to which the present derivatives of variant gp41peptides covalently bond may be either fixed or mobile. Fixed bloodcomponents are non-mobile blood components and include tissues, membranereceptors, interstitial proteins, fibrin proteins, collagens, platelets,endothelial cells, epithelial cells and their associated membrane andmembraneous receptors, somatic body cells, skeletal and smooth musclecells, neuronal components, osteocytes and osteoclasts, and all bodytissues especially those associated with the circulatory and lymphaticsystems. Mobile blood components are blood components that do not have afixed situs for any extended period of time, generally not exceeding 5minutes, and more usually one minute. These blood components are notmembrane-associated and are present in the blood for extended periods oftime in a minimum concentration of at least 0.1 μg/ml. Mobile bloodcomponents include serum albumin, transferrin, ferritin andimmunoglobulins such as IgM and IgG. The half-life of mobile bloodcomponents is at least about 12 hours. A carrier of choice for thisinvention is albumin conjugated through its free thiol.

In another embodiment of the invention is provided a method forgenerating peptide fusion inhibitors having anti-viral, virostatic oranti-fusogenic activity to prevent or treat infection by viruses,including retroviruses. According to the method, viral proteins involvedin viral entry into a cell and/or having fusogenic activity areidentified. The amino acid sequences of said viral proteins are thenscreened for alpha helix-forming regions believed to be involved inprotein-protein association. One of skill in the art can usecomputer-based algorithms to screen for alpha helix-forming regions ofprotein sequences. Computer-based algorithms useful for identifyingalpha helix-forming regions include, but are not limited to,Gamier-Robson and Chou-Fasman indices of helical preference, availablein such program suites as DNASTAR.

Peptides, derived from the alpha helix-forming regions of the viralproteins, can be designed according to the methods discussed supra bysubstituting predetermined amino acid residues with amino acid residuesthat enhance the hydrophilicity, hydrophobicity or alpha helix-formingtendencies of the peptide sequence. Alternatively, substitutions usingD-isomers of the naturally occurring L-amino acids or non-naturallyoccurring amino acids may be made to the peptides of the invention. Inone embodiment of the invention, at least two or more amino acidsubstitutions comprise D-isomers of the naturally occurring L-aminoacids. In another embodiment of the invention, the complete peptidesequence comprises D-isomers of the naturally occurring L-amino acids.Alterations such as these may serve to increase the stability,protease-resistance, activity, reactivity and/or solubility of thepeptides of the invention.

Derivatized forms of these peptides are useful as treatments havingextended half-lives once conjugated to blood components such as, forexample, serum albumin. Peptide sequences comprising D-isomers of thenaturally occurring L-amino acids are expected to demonstrate increasedresistance to protease activity in a manner proportional to the numberof D-isomers of the naturally occurring L-amino acids present in thepeptide sequence, independent of whether the peptides are conjugated toblood components.

This method of the invention further contemplates in vitro testing ofthe peptide compositions to verify anti-viral, virostatic oranti-fusogenic activity. For example, one of skill in the art couldmodify the teachings of Example 9 herein to similarly construct an assaythat screens for anti-viral activity. By way of a non-limiting example,one of skill in the art could utilize or modify the teachings of Example9 to test the effects of anti-viral peptides in the presence of a virushaving specificity for a cell type, such as for example, PBMCs, in orderto determine the IC₅₀ and TC₅₀ values. Following infection of a celltype in both the presence and absence of peptide inhibitors (withappropriate controls), and incubation of said cells, viral titers aredetermined and the IC₅₀ and TC₅₀ values determined.

Viruses to which this method of the invention is applicable include, butare not limited to, human retroviruses, including HIV-1 and HIV-2, humanT-lymphocyte viruses (HTLV-I and HTLV-II), and non-human retroviruses,including bovine leukosis virus, feline sarcoma virus, feline leukemiavirus, simian immunodeficiency virus (SIV), simian sarcoma virus, simianleukemia, and sheep progress pneumonia virus. Non-retroviral viruses mayalso be inhibited by the anti-viral, virostatic or anti-fusogenicpeptides, including but not limited to, human respiratory syncytialvirus (RSV), canine distemper virus, Newcastle disease virus, humanparainfluenza virus (HPV), influenza viruses, measles virus,Epstein-Barr viruses, hepatitis B viruses, and simian Mason-Pfizerviruses. Non-enveloped viruses may also be inhibited by the peptides ofthe invention, including but not limited to, picornaviruses such aspolio viruses, hepatitis A virus, enteroviruses, echoviruses, coxsachieviruses, papovaviruses such as papilloma virus, parvoviruses,adenoviruses, and reoviruses.

Peptide Synthesis

The derivatized variant gp41 peptides may be synthesized by standardmethods of solid phase peptide chemistry well known to any one ofordinary skill in the art. For example, the peptides may be synthesizedby solid phase chemistry techniques following the procedures describedby Steward et al. in Solid Phase Peptide Synthesis, 2nd Ed., PierceChemical Company, Rockford, Ill., (1984) using a Rainin PTI Symphonysynthesizer. Alternatively, peptides fragments may be synthesized andsubsequently combined or linked together to form the gp41 peptidesequences in solution (segment condensation, as described, for example,in U.S. Pat. No. 6,281,331 (the disclosures of both of which are hereinincorporated by reference)).

For solid phase peptide synthesis, a summary of the many techniques maybe found in Stewart et al. in “Solid Phase Peptide Synthesis”, W. H.Freeman Co. (San Francisco), 1963 and Meienhofer, Hormonal Proteins andPeptides, 1973, 2 46. For classical solution synthesis, see for exampleSchroder et al. in “The Peptides”, volume 1, Acacemic Press (New York).In general, such methods comprise the sequential addition of one or moreamino acids or suitably protected amino acids to a growing peptide chainon a polymer. Normally, either the amino or carboxyl group of the firstamino acid is protected by a suitable protecting group. The protectedand/or derivatized amino acid is then either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected and under conditions suitable for forming the amide linkage.The protecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is added, and soforth.

After all the desired amino acids have been linked in the propersequence, any remaining protecting groups (and any solid support) arecleaved sequentially or concurrently to yield the final peptide. Bysimple modification of this general procedure, it is possible to addmore than one amino acid at a time to a growing chain, for example, bycoupling (under conditions which do not racemize chiral centers) aprotected tripeptide with a properly protected dipeptide to form, afterdeprotection, a pentapeptide. Protective groups may be required duringthe synthesis process of the present peptide derivative. Theseprotective groups are conventional in the field of peptide synthesis,and can be generally described as chemical moieties capable ofprotecting the peptide derivative from reacting with other functionalgroups, Various protective groups are available commercially, andexamples thereof can be found in U.S. Pat. No. 5,493,007, which isherein incorporated by reference. Typical examples of suitableprotective groups include acetyl, fluorenylmethyloxycarbonyl (FMOC),t-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), etc. In addition,Table 7 provides both the three letter and one letter abbreviations ofthe naturally occurring amino acids.

TABLE 7 Naturally Occurring Amino Acids and Their Abbreviations Name3-letter abbreviation 1-letter abbreviation Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

A particularly preferred method of preparing the variant gp41 peptidesinvolves solid phase peptide synthesis wherein the amino acidα-N-terminal is protected by an acid or base sensitive group. Suchprotecting groups should have the properties of being stable to theconditions of peptide linkage formation while being readily removablewithout destruction of the growing peptide chain or racemization of anyof the chiral centers contained therein. Examples of N-protecting groupsand carboxy-protecting groups are disclosed in Greene, “ProtectiveGroups In Organic Synthesis,” (John Wiley & Sons, New York pp. 152-186(1981)), which is herein incorporated by reference. Examples ofN-protecting groups comprise, without limitation, loweralkanoyl groupssuch as formyl, acetyl (“Ac”), propionyl, pivaloyl, t-butylacetyl andthe like; other acyl groups include 2-chloroacetyl, 2-bromoacetyl,trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyland the like; sulfonyl groups such as benzenesulfonyl,p-toluenesulfonyl, o-nitrophenylsulfonyl,2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), and the like; carbamateforming groups such as t-amyloxycarbonyl, benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl (boc), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl(Aloe), 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl,4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl,isobornyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl,cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; arylalkyl groupssuch as benzyl, biphenylisopropyloxycarbonyl, triphenylmethyl,benzyloxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like andsilyl groups such as trimethylsilyl and the like. Preferredα-N-protecting group are o-nitrophenylsulfenyl;9-fluorenylmethyloxycarbonyl; t-butyloxycarbonyl (boc),isobornyloxycarbonyl; 3,5-dimethoxybenzyloxycarbonyl; t-amyloxycarbonyl;2-cyano-t-butyloxycarbonyl, and the like, 9-fluorenyl-methyloxycarbonyl(Fmoc) being more preferred, while preferred side chain N-protectinggroups comprise 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro,p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl for side chain amino groups like lysine andarginine; Aloe for lysine; benzyl, o-bromobenzyloxycarbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac) for tyrosine; t-butyl, benzyl and tetrahydropyranyl forserine; trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl forhistidine; formyl for tryptophan; benzyl and t-butyl for aspartic acidand glutamic acid; and triphenylmethyl (trityl) for cysteine.

A carboxy-protecting group conventionally refers to a carboxylic acidprotecting ester or amide group. Such carboxy protecting groups are wellknown to those skilled in the art, having been extensively used in theprotection of carboxyl groups in the penicillin and cephalosporin fieldsas described in U.S. Pat. Nos. 3,840,556 and 3,719,667, the disclosuresof which are herein incorporated by reference.

Representative carboxy protecting groups comprise, without limitation,C1-C8 loweralkyl; arylalkyl such as phenethyl or benzyl and substitutedderivatives thereof such as alkoxybenzyl or nitrobenzyl groups;arylalkenyl such as phenylethenyl; aryl and substituted derivativesthereof such as 5-indanyl; dialkylaminoalkyl such as dimethylaminoethyl;alkanoyloxyalkyl groups such as acetoxymethyl, butyryloxymethyl,valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl,1-(propionyloxy)-1-ethyl, 1-(pivaloyloxyl)-1-ethyl,1-methyl-1-(propionyloxy)-1-ethyl, pivaloyloxymethyl,propionyloxymethyl; cycloalkanoyloxyalkyl groups such ascyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxy-methyl;aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl;arylalkylcarbonyloxyalkyl such as benzykarbonyloxymethyl,2-benzylcarbonyloxyethyl; alkoxycarbonylalkyl orcycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl;alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, t-butyloxycarbonyl-oxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl;aryloxy-carbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)-ethyl; alkoxyalkylcarbonyloxyalkyl such as2-(1-methoxy-2-methylpropan-2-oyloxy)-ethyl;arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl;arylalkenyloxycarbonyloxyalkyl such as2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl; alkoxycarbonylaminoalkylsuch as t-butyloxycarbonylaminomethyl; alkylaminocarbonyl-aminoalkylsuch as methylaminocarbonylaminomethyl; alkanoylaminoalkyl such asacetylaminomethyl; heterocycliccarbonyloxyalkyl such as4-methylpiperazinyl-carbonyloxymethyl; dialkylaminocarbonylalkyl such asdimethylaminocarbonylmethyl, diethylaminocarbonylmethyl;(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl; and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl. Representative amide carboxyprotecting groups comprise, without limitation, aminocarbonyl andloweralkylaminocarbonyl groups. Of the above carboxy-protecting groups,loweralkyl, cycloalkyl or arylalkyl ester, for example, methyl ester,ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butylester, isobutyl ester, amyl ester, isoamyl ester, octyl ester,cyclohexyl ester, phenylethyl ester and the like or an alkanoyloxyalkyl,cycloalkanoyloxyalkyl, aroyloxyalkyl or an arylalkylcarbonyloxyalkylester are preferred. Preferred amide carboxy protecting groups areloweralkylaminocarbonyl groups.

In the solid phase peptide synthesis method, the α-C-terminal amino acidis attached to a suitable solid support or resin. Suitable solidsupports useful for the above synthesis are those materials that areinert to the reagents and reaction conditions of the stepwisecondensation-deprotection reactions, as well as being insoluble in themedia used. The preferred solid support for synthesis of α-C-terminalcarboxy peptides is4-hydroxymethylphenoxyacetyl-4′-methylbenzyhydrylamine resin (HMPresin). The preferred solid support for α-C-terminal amide peptides isan Fmoc-protected Ramage resin, manufactured and sold by Bachem Inc.,California.

In preferred syntheses, the linking lysine is protected by Aloc. Afterthe synthesis is complete, the Aloe is cleaved by Pd(Ph₃)₄ while thepeptide is still on the resin, and allows the coupling of the linkermolecule and the maleimide group. Specifically, the linker is[2-(2-amino)ethoxyl]ethoxy acetic acid, and the maleimide group is3′-maleimidopropionic acid. After the modification, the Fmoc groups areremoved and the peptide is cleaved off the resin.

At the end of the solid phase synthesis, the peptide is removed from theresin and deprotected, either in successive operations or in a singleoperation. Removal of the peptide and deprotection can be accomplishedconventionally in a single operation by treating the resin-boundpolypeptide with a cleavage reagent comprising thioanisole, triisopropylsilane, phenol, and trifluoroacetic acid. In cases wherein theα-C-terminus of the peptide is an alkylamide, the resin is cleaved byaminolysis with an alkylamine. Alternatively, the peptide may be removedby transesterification, e.g. with methanol, followed by aminolysis or bydirect transamidation. The protected peptide may be purified at thispoint or taken to the next step directly. The removal of the side chainprotecting groups is accomplished using the cleavage mixture describedabove. The fully deprotected peptide can be purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin (acetate form); hydrophobic adsorptionchromatography on underivatized polystyrene-divinylbenzene (such asAmberlite XAD); silica gel adsorption chromatography; ion exchangechromatography on carboxymethylcellulose; partition chromatography, e.g.on Sephadex G-25, LH-20 or countercurrent distribution; high performanceliquid chromatography (HPLC), especially reverse-phase HPLC on octyl- orphenyl/hexylsilyl-silica bonded phase column packing. The skilledartisan can determine the preferred chromatographic steps or sequencesrequired to obtain acceptable purification of the variant gp41 peptides.

Alternatively, peptide fragments, including addition of the maleimidegroup can be synthesized in solid phase, and the final derivatizedpeptide can be obtained by solution coupling of these fragments.

Molecular weights of these peptides may be determined using Electrospraymass spectroscopy or MALDI-TOF mass spectroscopy.

Therapeutic Use of the Modified Peptides

The variant gp41 peptides, including compounds listed in Tables 1, 2 and3 and FIG. 1, inhibit viral infection of cells, for example, byinhibiting cell-cell fusion or free virus infection. The route ofinfection may involve membrane fusion, as occurs in the case ofenveloped or encapsulated viruses, or some other fusion event involvingviral and cellular structures such as cellular receptors.

The variant gp41 peptides may be administered in vivo such thatconjugation with blood components occurs in vivo, or they may be firstconjugated to blood components ex vivo and the resulting conjugatedderivative administered in vivo. In another embodiment of the invention,plasmaphoresis is utilized to separate desired blood components in apatient's blood sample, which are then conjugated to the peptides of theinvention prior to administration back to the patient.

Thiol groups are less abundant in vivo than, for example, amino groupsin plasma proteins. Hence the maleimide-modified variant gp41 peptide(s)will covalently bond to fewer proteins. For example, in albumin (themost abundant blood protein) there is only one thiol group. Thus, amodified gp41 peptide-maleimide-albumin conjugate will tend to compriseapproximately a 1:1 molar ratio of gp41 peptide to albumin. In additionto albumin, IgG molecules (class II) also have free thiols. Since IgGmolecules and serum albumin make up the majority of the soluble proteinin blood they also make up the majority of the free thiol groups inblood that are available to covalently bond to the variant gp41peptides.

Further, even among free thiol-containing blood proteins, includingIgGs, specific labeling with a maleimide leads to the preferentialformation of a modified gp41 peptide-maleimide-albumin conjugate due tothe unique characteristics of albumin itself. The single free thiolgroup of albumin, highly conserved among species, is located at aminoacid residue 34 (Cys34). It has been demonstrated recently that theCys34 of albumin has increased reactivity relative to free thiols onother free thiol-containing proteins. This is due in part to the verylow pK value of 5.5 for the Cys34 of albumin. This is much lower thantypical pK values for cysteine residues in general, which are typicallyabout 8. Due to this low pK, under normal physiological conditions Cys34of albumin is predominantly in the ionized form, which dramaticallyincreases its reactivity. In addition to the low pK value of Cys34,another factor which enhances the reactivity of Cys34 is its location,which is in a hydrophobic pocket close to the surface of one loop ofregion V of albumin. This location makes Cys34 readily available toligands of all kinds, and is an important factor in Cys34's biologicalrole as free radical trap and free thiol scavenger. These propertiesmake Cys34 highly reactive with gp41 peptides harboring maleimidelinkages, and the reaction rate acceleration can be as much as 1000-foldrelative to rates of reaction of variant gp41 peptides with maleimidelinkages with other free-thiol containing proteins.

Another advantage of modified gp41 peptide-maleimide-albumin conjugatesis the reproducibility associated with the 1:1 loading of peptide toalbumin specifically at Cys34. Other techniques, such as glutaraldehyde,DCC, EDC and other chemical activations of, e.g, free amines, lack thisselectivity. For example, albumin contains 52 lysine residues, 25-30 ofwhich are located on the surface of albumin and therefore accessible forconjugation. Activating these lysine residues, or alternativelymodifying variant gp41 peptides to couple through these lysine residues,results in a heterogenous population of conjugates. Even if 1:1 molarratios of gp41 maleimide peptides to albumin are employed, the yield ofamine derivatized albumin will consist of multiple conjugation products,some containing 0, 1, 2 or more gp41 peptides per albumin, and eachhaving the peptide randomly coupled at any one or more of the 25-30available lysine sites. Given the numerous possible combinations,characterization of the exact composition and nature of each conjugatebatch becomes difficult, and batch-to-batch reproducibility is all butimpossible, making such conjugates less desirable as a therapeutic.

Additionally, while it would seem that conjugation through lysineresidues of albumin would at least have the advantage of delivering moretherapeutic agent per albumin molecule, studies have shown that a 1:1ratio of therapeutic agent to albumin is preferred. In an article byStehle, et al., “The Loading Rate Determines Tumor Targeting propertiesof Methotrexate-Albumin Conjugates in Rats,”Anti-Cancer Drugs, Vol. 8,pp. 677-685 (1988), (incorporated herein by reference in its entirety),the authors report that a 1:1 ratio of the anti-cancer drug methotrexateto albumin conjugated via glutaraldehyde gave the most promisingresults. These conjugates were preferentially taken up by tumor cells,whereas conjugates bearing 5:1 to 20:1 methotrexate molecules hadaltered HPLC profiles and were quickly taken up by the liver in vivo. Itis postulated that at these higher ratios, conformational changes toalbumin diminish its effectiveness as a therapeutic carrier.

Through controlled administration of the variant gp41 peptides in vivo,one can control the specific labeling of albumin and IgG in vivo. Intypical administrations, 80-90% of the administered derivatized variantgp41 peptides will label albumin and less than 5% will label IgG. Tracelabeling of free thiols such as glutathione will also occur. Suchspecific labeling is preferred for in vivo use as it permits an accuratecalculation of the estimated half-life of the variant gp41 peptides.

In addition to providing controlled specific in vivo labeling, thederivatized variant gp41 peptides can provide specific labeling of serumalbumin and IgG ex vivo. Such ex vivo labeling involves the addition ofthe variant gp41 peptides harboring maleimide linkages to blood, serumor saline solution containing serum albumin and/or IgG. Once conjugationhas occurred ex vivo with the variant gp41 peptides, the blood, serum orsaline solution can be readministered to the patient's blood for in vivotreatment, or lyophilized.

Variant gp41 peptides may be used alone or in combination to optimizetheir therapeutic effects. In another embodiment of the invention,variant gp41 peptides are co-administered with one or more additionalantiviral HIV treatments. Additional antiviral HIV treatments that canbe co-administered with the variant gp41 peptides include, but are notlimited to, AGENERASE (amprenavir; GlaxoSmithKline); COMBIVIR(lamivudine, zidovudine; GlaxoSmithKline); CRIXIVAN (indinavir, IDV,MK-639; Merck); EMTRIVA (FTC, emtricitabine; Gilead Sciences); EPIVIR(lamivudine, 3TC; GlaxoSmithKline); FORTOVASE (saquinavir; Hoffmann-LaRoche); HIVID (Zalcitabine, ddC, dideoxycytidine; Hoffmann-La Roche);INVIRASE (saquinavir mesylate, SQV; Hoffmann-La Roche); KALETRA(lopinavir, ritonavir; Abbott Laboratories); NORVIR (ritonavir, ABT-538;Abbott Laboratories); RESCRIPTOR (Delaviridine, DLV; Pfizer); RETROVIR(zidovudine, AZT, azidothymidine, ZDV; GlaxoSmithKline); REYATAZ(atazanavir sulfate; Bristol Myers-Squibb); SUSTIVA (efavirenz; BristolMyers-Squibb); TRIZIVIR (abacavir, zidovudine, lamivudine;GlaxoSmithKline); VIDEX EC (enteric coated didanosine; BristolMyers-Squibb); VIDEX (didanosine, ddl, dideoxyinosine; BristolMyers-Squibb); VIRACEPT (nelfinavir mesylate, NFV; AgouronPharmaceuticals); VIRAMUNE (nevirapine, BI-RG-587; BoehringerIngelheim); VIREAD (tenofovir disoproxil fumarate; Gilead); ZERIT(stavudine, d4T; Bristol Myers-Squibb); ZIAGEN (abacavir;GlaxoSmithKline).

In an additional embodiment of the invention, variant gp41 peptides areco-administered with one or more additional compounds used to treat HIVor HIV-induced diseases. These additional compounds that can beco-administered with the variant gp41 peptides include, but are notlimited to, TRIMETREXATE GLUCURONATE (for the treatment of Pneumocystiscarinii pneumonia); GANCICLOVIR (for the treatment of cytomegalovirusretinitis); aerosolized PENTAMIDINE (for the treatment of Pneumocystiscarinii pneumonia); ERYTHROPOIETIN (for the treatment ofZidovudine-related anemia); ATOVAQUONE (for the treatment ofPneumocystis carinii pneumonia); RIFABUTIN (for the treatment ofMycobacterium avium); VISTIDE (for the treatment of relapsingcytomegalovirus retinitis); and SEROSTIM (for the treatment ofAIDS-related wasting).

Variant gp41 peptides, including but not limited to those peptidesprovided in Tables 1, 2 and 3, as well as FIG. 1, can be co-administeredwith one or more additional variant gp41 peptides listed in Tables 1, 2and 3, as well as FIG. 1. In another embodiment of the invention,variant gp41 peptides, including but not limited to those peptidesprovided in Tables 1, 2 and 3, as well as FIG. 1, can be co-administeredwith T-20 or T-1249 peptides.

Variant gp41 peptides are administered in a physiologically acceptablemedium, e.g. deionized water, phosphate buffered saline (PBS), saline,aqueous ethanol or other alcohol, plasma, proteinaceous solutions,mannitol, aqueous glucose, alcohol, vegetable oil, or the like.Preferably the pharmaceutical composition comprising the variant gp41peptides is administered with a pharmaceutically acceptable carrier.Other components which may be added include buffers, where the media aregenerally buffered at a pH in the range of about 5 to 10, where thebuffer will generally range in concentration from about 50 to 250 mM;salt, where the concentration of salt will generally range from about 5to 500 mM; physiologically acceptable stabilizers, and the like. Thecompositions may be lyophilized for convenient storage and transport.

Variant gp41 peptides may be administered orally, parenterally, such asintravascularly (IV), intraarterially (IA), intramuscularly (IM),subcutaneously (SC), or the like. Administration may in appropriatesituations be by transfusion. In some instances, where reaction of thefunctional group is relatively slow, administration may be oral, nasal,rectal, transdermal or by aerosol means, where the nature of theconjugate allows for transfer to the vascular system. Usually a singleinjection will be employed although more than one injection may be used,if desired. The peptide derivative may be administered by any convenientmeans, including syringe, trocar, catheter, or the like. The particularmanner of administration will vary depending upon the amount to beadministered, whether a single bolus or continuous administration, orthe like. Preferably, the administration will be intravascularly, wherethe site of introduction is not critical to this invention, preferablyat a site where there is rapid blood flow, e.g., intravenously,peripheral or central vein. Other routes may find use where theadministration is coupled with slow release techniques or a protectivematrix. The intent is that the variant gp41 peptides be effectivelydistributed in the blood, so as to be able to react with the bloodcomponents. The amount of the conjugate administered will vary widely,generally ranging from about 1 mg to 500 mg. The total administeredintravascularly will generally be in the range of about 0.5 μg/kg bodyweight to about 50 mg/kg, more usually about 0.5 mg/kg to about 10mg/kg.

By bonding to long-lived components of the blood, such asimmunoglobulin, serum albumin, red blood cells and platelets, a numberof advantages ensue. The activity of the variant gp41 peptides isextended for days to weeks. Only one administration needs to be givenduring this period of time. Greater specificity can be achieved, sincethe active compound will be primarily bound to large molecules where itis less likely to be taken up intracellularly and interfere with otherphysiological processes.

The formation of the covalent bond with the blood component may occur invivo or ex vivo. For ex vivo covalent bond formation, derivatizedvariant gp41 peptides are added to blood serum or a saline solutioncontaining purified blood components such as human serum albumin or IgG,to permit covalent bond formation between the derivative and the bloodcomponent. In a preferred embodiment, the variant gp41 peptides arereacted with human serum albumin in saline solution. After formation ofthe conjugate, the latter may be administered to the subject orlyophilized.

The blood of the mammalian host may be monitored for the activity and/orthe presence of the variant gp41 peptides. By taking a blood sample fromthe host at different times, one may determine whether variant gp41peptides have become bonded to the long-lived blood components insufficient amount to be therapeutically active and, thereafter, thelevel of the variant gp41 peptides in the blood. If desired, one mayalso determine to which of the blood components variant gp41 peptidesare covalently bonded. Monitoring may also take place by using assaysspecific for gp41 peptide activity, HPLC-MS or antibodies directedagainst variant gp41 peptides.

The variant gp41 peptides can be administered to patients according tothe methods described herein and other methods known in the art.Patients for whom therapy is contemplated include patients infected withany of the viruses referred to herein, particularly HIV-1 and HIV-2.Effective therapeutic dosages of the variant gp41 peptides may bedetermined through procedures well known by those in the art and willtake into consideration any concerns regarding potential toxicity ofthese gp41 peptides.

The variant gp41 peptides can also be administered prophylactically topreviously uninfected individuals. This administration can beadvantageous in cases where an individual has been subjected to a highrisk of exposure to a virus, as can occur when a patient has been incontact with an infected individual and there is a high risk of viraltransmission. This can be especially advantageous where there is noknown cure for the virus, such as the HIV virus. By way of anon-limiting example, prophylactic administration of a gp41 peptidewould be advantageous in a situation where a health care worker has beenexposed to blood from an HIV-infected individual, or in other situationswhere patients have engaged in high-risk activities that potentiallyexpose those individuals to the HIV virus. Other applications of thevariant gp41 peptides encompass administration of the same toindividuals harboring a virus, such as HIV, in order to prevent thetransmission of the virus from the infected individual to a non-infectedindividual. Such applications also include the prevention of mother toinfant transmission by breast feeding or other daily contacts, ortransmission occurring through sexual activity.

In another embodiment of the invention, variant gp41 peptides, includingbut not limited to those peptides provided in Tables 1, 2 and 3, as wellas FIG. 1, can be co-administered with one or more additional peptideslisted in Tables 1, 2 and 3, FIG. 1, T-20, T-1249, or other HIVtreatments to prevent the replication of HIV (including HIV-1, HIV-2, orall other serotypes thereof) and SIV viral particles in the patient.

Topical Application

The variant gp41 peptides, including those provided in Tables 1, 2 and 3and FIG. 1 can be used alone or in the form of a composition containingor consisting essentially of an effective concentration of the peptideand a pharmaceutically acceptable carrier. An effective concentrationcan be determined by observing whether virus infection can be impededupon application of the agent(s).

The compositions of the invention include topical microbicidal,virostatic or anti-fusogenic uses for both in vitro and in vivopurposes, especially for intravaginal and intrarectal use. For thesepurposes the modified peptide can be formulated in any appropriatevehicle, provided, that is, that the anti-fusion activity of themodified peptide is not diminished by the vehicle. Thus, thecompositions can be in the form of creams, gels, foams, lotions,ointments, tablets, solutions or sprays. The carrier or vehicle diluentcan be aqueous or non-aqueous, for example alcoholic or oleaginous, or amixture thereof, and may additionally contain other surfactants,emollients, lubricants, stabilizers, dyes, perfumes, antimicrobialagents either as active ingredients or as preservatives, and acids orbases for adjustment of pH. The preferred pH is about 4 to 5.Conventional methods are used in preparing the compositions.

Preferably, the pharmaceutically acceptable carrier or vehicle fortopically applied compositions is in the form of a liquid, jelly, orfoam containing the compound of this invention. The compound can beincorporated into: (a) ointments and jellies, (b) inserts(suppositories, sponges, and the like), (c) foams, (d) douches and (e)cleansing fluids or body washes. The composition is preferablyintroduced into the vagina of a female or the rectum of a male orfemale, at about the time of, and preferably prior to, sexualintercourse, but may also be administered to other mucous membranes. Thecompositions can be employed for the treatment of and for protectionagainst, sexually transmitted diseases including HIV. The manner ofadministration will preferably be designed to obtain direct contact ofthe peptide-containing compositions of the invention with the causativeagents of sexually transmitted diseases.

For topical applications, the pharmaceutically acceptable carrier mayadditionally comprise organic solvents, emulsifiers, gelling agents,moisturizers, stabilizers, other surfactants, wetting agents,preservatives, time release agents, and minor amounts of humectants,sequestering agents, dyes, perfumes, and other components commonlyemployed in pharmaceutical compositions for topical administration.

With regard to the articles provided by the present invention, thecompositions of the invention may be impregnated into absorptivesubstrate materials, such as sponges, or coated onto the surface ofsolid substrate materials, such as condoms, diaphragms or medicalgloves, to deliver the compositions to vaginal or other potentiallyinfectable epithelium, preferably before or during sexual intercourse.Other articles and delivery systems of this type will be readilyapparent to those skilled in the art. The presently preferred articlesare condoms, which are coated by spraying modified peptides onto thesurfaces of the condoms, or by impregnating the peptides into the condomduring manufacture by processes known in the art. Preferred coatingcompositions include silicon which provides lubricity and releases themodified peptide in a time release manner. Bioadhesive polymers may alsobe used to prolong the time release aspects of the particular topical orother medicament employed.

Solid dosage forms for topical administration include suppositories,powders, tablets, and granules. In solid dosage forms, the compositionsmay be admixed with at least one inert diluent such as sucrose, lactose,or starch, and may additionally comprise lubricating agents, bufferingagents and other components well known to those skilled in the art.

Actual dosage levels of the modified peptides in the compositions andarticles of the invention may be varied so as to obtain amounts at thesite of sexually transmitted fluids to obtain the desired therapeutic orprophylactic response for a particular peptide and method ofadministration. Accordingly, the selected dosage level will depend onthe nature and site of infection, the desired therapeutic response, theroute of administration, the desired duration of treatment and otherfactors. Generally, the preferred dosage for modified peptides of thisinvention will be in the range of about 0.01 to 2.0 wt. percent. Apreferred topical vaginal dosage form is a cream or suppository asdescribed above containing from 0.01 to 2.0 wt. percent of thecomposition according to the invention. In each treatment, typicallytwice daily, from about 1 to about 5 ml of such dosage form is appliedintravaginally, preferably high in the vaginal orifice or into therectum. Greater amounts are generally avoided to minimize leakage.

The methods and compositions of the invention can be used to prevent andtreat a broad spectrum of infections by pathogenic microbes.

EXAMPLES

In order to facilitate a more complete understanding of the invention, anumber of Examples are provided below. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

General

Unless stated otherwise, the synthesis of each variant gp41 peptide wasperformed using an automated solid-phase procedure on a Symphony PeptideSynthesizer with manual intervention during the generation of thederivative. The synthesis was performed on Fmoc-protected Ramage amidelinker resin, using Fmoc-protected amino acids. Coupling was achieved byusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) as activator in N,N-dimethylformamide (DMF)solution and diisopropylethylamine (DIEA) as base. The Fmoc protectivegroup was removed using 20% piperidine/DMF. All amino acids used duringthe synthesis possess the L-stereochemistry. Glass reaction vessels wereused during the synthesis.

Example 1 Synthesis of FB005

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Glu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr-OH, Fmoc-Met-OH, Fmoc-Asn-OH,Fmoc-Asn-OH, Fmoc-Trp-OH, Fmoc-Ile-OH, Fmoc-Gln-OH, Fmoc-Glu-OH,Fmoc-Leu-OH, Fmoc-Ser-OH. They were dissolved in N,N-dimethylformamide(DMF) and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid was acetylated using acetic acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA).

Step 2: The peptide was cleaved from the resin using 85% TFA/5%triisopropylsilane (TIPS)/5% a thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et2O (Step 2).

Example 2 Synthesis of FB005M

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr-OH, Fmoc-Met-OH, Fmoc-Asn-OH,Fmoc-Asn-OH, Fmoc-Trp-OH, Fmoc-Ile-OH, Fmoc-Gln-OH, Fmoc-Glu-OH,Fmoc-Leu-OH, Fmoc-Ser-OH. They were dissolved in N,N-dimethylformamide(DMF) and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid was acetylated using acetic acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA).

Step 2: The selective deprotection of the Lys (Aloe) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of C6H6 CHCl3 (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl3 (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of theFmoc-AEEA-OH and the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol (iPrOH).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt2O (Step 4).

Example 3 Synthesis of FB005CM

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-His-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Glu-OH,Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu-OH, Fmoc-Trp(Boc)-OH,Fmoc-Thr-OH, Fmoc-Met-OH, Fmoc-Asn-OH, Fmoc-Asn-OH, Fmoc-Trp-OH,Fmoc-Ile-OH, Fmoc-Gln-OH, Fmoc-Glu-OH, Fmoc-Leu-OH, Fmoc-Ser-OH. Theywere dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid was acetylated using acetic acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uranium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA).

Step 2: The selective deprotection of the Lys (Aloe) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of C6H6 CHCl3 (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl3 (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of theFmoc-AEEA-OH and the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol (iPrOH).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt2O (Step 4).

Example 4 Synthesis of FB006

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Trp(Boc)-OH. They were dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid was acetylated using acetic acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt2O (Step 4).

Example 5 Synthesis of FB006M

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH. They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (WV)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Theamino group of the final amino acid was acetylated using acetic acidactivated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of C6H6 CHCl3 (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl3 (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of theFmoc-AEEA-OH and the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol (iPrOH).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt2O (Step 4).

Example 6 Synthesis of FB007M

Step 1: The example describes the solid phase peptide synthesis of thecompound on a mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-His-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Trp(Boc)-OH. They were dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid was acetylated using acetic acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA).

Step 2: The selective deprotection of the Lys (Aloe) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of C6H6 CHCl3 (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl3 (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of theFmoc-AEEA-OH and the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol (iPrOH).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEta) (Step 4).

Example 7 Synthesis of FB010M

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gin(Trt)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Trp(Boc)-OH. They were dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid was acetylated using acetic acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and diisopropylethylamine (DIEA).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of C6H6 CHCl3 (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl3 (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5mL).

Step 3: The synthesis was then re-automated for the addition of theFmoc-AEEA-OH and the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol (iPrOH).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt2O (Step 4).

Example 8 Synthesis of FB010KM

Step 1: The example describes the solid phase peptide synthesis of thecompound on a 1 mmole scale. The following protected amino acids weresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Phe-OH,Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH. They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Theamino group of the final amino acid was acetylated using acetic acidactivated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).

Step 2: The selective deprotection of the Lys (Aloe) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of C6H6 CHCl3 (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl3 (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of theFmoc-AEEA-OH and the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol (iPrOH).

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt2O (Step 4).

Example 9 Viral Inhibition by Modified Peptides

The antiviral activity and cytotoxicity of FB005, FB006, FB006M, FB007M,FB010KM, and FM010M were tested against HIV-1_(IIIB) in fresh human PBMCcultures. The four modified peptides FB006M, FB007M, FB010M, and FB010KMwere conjugated to human serum albumin (HSA) by mixing prior to theantiviral test. The results appear in Table 8 below, where IC₅₀ value isthe 50% viral inhibition drug concentration, and TC₅₀ value is the 50%cytotoxicity drug concentration.

Cellular Anti-HIV Assay

Pretitered aliquots of HIV-1_(IIIB) was removed from the freezer (−80°C.) and thawed rapidly to room temperature in a biological safetycabinet immediately before use.

Fresh human PBMCs were isolated from screened donors, seronegative forHIV and HBV (Interstate Blood Bank, Inc.; Memphis, Tenn.). Cells werepelleted/washed 2-3 times by low speed centrifugation and resuspensionin PBS to remove contaminating platelets. The Leukophoresed blood wasthen diluted 1:1 with Dulbecco's phosphate buffered saline (PBS) andlayered over 14 mL of Lymphocyte Separation Medium in a 50 mL centrifugetube and then centrifuged for 30 minutes at 600×g. Banded PBMCs weregently aspirated from the resulting interface and subsequently washed 2×with PBS by low speed centrifugation. After the final wash, cells wereenumerated by trypan blue exclusion and re-suspended at 1×10⁷ cells/mLin RPMI 1640 supplemented with 15% Fetal Bovine Serum (FBS), 2 mML-glutamine, 4 μg/mL Phytohemagglutinin (PHA-P, Sigma). The cells wereallowed to incubate for 48-72 hours at 37° C. After incubation, PBMCswere centrifuged and resuspended in RPMI 1640 with 15% FBS, 2 mML-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mLgentamycin, and 20 U/mL recombinant human IL-2 (R&D Systems, Inc). PBMCswere maintained in this medium at a concentration of 1-2×10⁶ cells/mLwith biweekly medium changes until used in the assay protocol. Cellswere kept in culture for a maximum of two weeks before being deemed tooold for use in assays and discarded. Monocytes were depleted from theculture as the result of adherence to the tissue culture flask.

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled, diluted in fresh medium to a finalconcentration of 1×10⁶ cells/mL, and plated in the interior wells of a96 well round bottom microplate at 50 μL/well (5×10⁴ cells/well). Eachplate contains virus/cell control wells (cells plus virus), experimentalwells (drug plus cells plus virus) and compound control wells (drug plusmedia without cells, necessary for MTS monitoring of cytotoxicity).Since HIV-1 is not cytopathic to PBMCs, this allows the use of the sameassay plate for both antiviral activity and cytotoxicity measurements.Test drug dilutions were prepared at a 2× concentration in microtitertubes and 100 μL of each concentration was placed in appropriate wellsusing the standard format. 50 μL of a predetermined dilution of virusstock was placed in each test well (final MOI≈0.1). The PBMC cultureswere maintained for seven days following infection at 37° C., 5% CO₂,after which cell-free supernatant samples were collected for analysis ofreverse transcriptase activity and/or HIV p24 content. Following removalof supernatant samples, compound cytotoxicity was measured by additionof MTS to the plates for determination of cell viability. Wells werealso examined microscopically and any abnormalities noted.

Secondary Cytotoxicity Assay

In order to test the cytotoxicity of the compounds at higherconcentrations than those used in the anti-HIV efficacy evaluation, asecondary assay was used. This assay was essentially the same asdescribed above for the anti-HIV efficacy evaluation, however no viruswas added to the wells (replaced by media without virus) and thehigh-test concentration was increased to 25 μM. Following incubation,plates were assayed for cell viability using MTS as described below.

TABLE 8 Compound Comment IC₅₀ (nM) TC₅₀ (nM) FB005 Unmodified peptide0.93 14,300 FB006 Unmodified peptide 1.41 15,900 FB006M modified peptide3.94 >25,000 conjugated with HSA FB007M modified peptide 9.09 >25,000conjugated with HSA FB010M modified peptide 7.78 >25,000 conjugated withHSA FB010KM modified peptide 15.7 >25,000 conjugated with HSA

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. Modificationsof the above-described modes for carrying out the invention that areobvious to persons of skill in medicine, immunology, virology,pharmacology, protein synthesis and modification and/or related fieldsare intended to be within the scope of the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All such publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Certain peptides and derivatives thereof that are useful in preventingand/or treating viral infection, particularly HIV infection, weredisclosed in U.S. Provisional Patent Application No. 60/412,797, filedSep. 24, 2002, the contents of which (including any sequences containedtherein) is herein incorporated by reference in its entirety.

1-36. (canceled)
 37. An isolated, modified peptide comprising the aminoacid sequence of SEQ ID NO:1; SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7,and having at least one substituted amino acid residue at apredetermined position in the peptide sequence, wherein the at least onesubstituted amino acid residue is a hydrophilic amino acid residue, ahydrophobic amino acid residue, an amino acid residue having apropensity to form alpha helices, a D-isomer of one of the naturallyoccurring L-amino acids, or a non-naturally occurring amino acidresidue.
 38. An isolated peptide derivatized with a maleimide linkingmoiety comprising the amino acid sequence of SEQ ID NO:3; SEQ ID NO:12,or SEQ ID NO:13.
 39. The derivatized peptide of claim 38, wherein themaleimido group is 3′-maleimidopropionate connected to the epsilon aminogroup of lysine by [2-(2-amino)ethoxyl]ethoxy acetic acid.
 40. Apharmaceutical composition comprising the peptide of claim
 37. 41. Apharmaceutical composition comprising the derivatized peptide of claim38.
 42. A conjugate comprising the derivatized peptide of claim 37conjugated to a blood component.
 43. The conjugate of claim 42, whereinthe blood component is human serum albumin protein, human transferrinprotein, human ferritin protein, human immunoglobulin proteins, humanferritin protein, human α-2-macroglobulin protein, human thyroxinbinding protein, human steroid binding proteins, or a combinationthereof.
 44. A conjugate comprising the derivatized peptide of claim 38conjugated to a blood component.
 45. The conjugate of claim 44, whereinthe blood component is human serum albumin protein, human transferrinprotein, human ferritin protein, human immunoglobulin proteins, humanferritin protein, human α-2-macroglobulin protein, human thyroxinbinding protein, human steroid binding proteins, or a combinationthereof.
 46. A method of inhibiting HIV-1 viral replication in a hostcomprising administrating the peptide of claim
 37. 47. The method ofclaim 46, wherein the peptide is administered orally, topically,intravascularly, intraarterially, intramuscularly, or subcutaneously.48. The method of claim 46, wherein the peptide is co-administered withone or more additional HIV treatment(s).
 49. The method of claim 46,wherein the said one or more additional HIV treatment(s) comprises atleast one other variant gp41 peptide.
 50. The method of claim 49,wherein the additional HIV treatment(s) is selected from the groupconsisting of: AGENERASE, COMBIVIR, CRIXIVAN, EMTRIVA, EPIVIR,FORTOVASE, HIVID, INVIRASE, KALETRA, NORVIR, RESCRIPTOR, RETROVIR,REYATAZ, SUSTIVA, TRIZIVIR, VIDEX EC, VIDEX, VIRACEPT, VIRAMUNE, VIREAD,ZERIT, ZIAGEN, or combinations thereof.
 51. A method of inhibiting HIV-1viral replication in a host comprising administrating the derivatizedpeptide of claim
 38. 52. The method of claim 51, wherein the derivatizedpeptide is administered orally, topically, intravascularly,intraarterially, intramuscularly, or subcutaneously.
 53. The method ofclaim 52, wherein the derivatized peptide is co-administered with one ormore additional HIV treatment(s).
 54. The method of claim 53, whereinthe said one or more additional HIV treatment(s) comprises at least oneother variant gp41 peptide.
 55. The method of claim 54, wherein theadditional HIV treatment(s) is selected from the group consisting of:AGENERASE, COMBIVIR, CRIXIVAN, EMTRIVA, EPIVIR, FORTOVASE, HIVID,INVIRASE, KALETRA, NORVIR, RESCRIPTOR, RETROVIR, REYATAZ, SUSTIVA,TRIZIVIR, VIDEX EC, VIDEX, VIRACEPT, VIRAMUNE, VIREAD, ZERIT, ZIAGEN, orcombinations thereof.